The term colony stimulating factor (CSF) is inclusive of granulocyte/macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF) and granulocyte-colony stimulating factor (G-CSF), which are produced by T-cells, macrophages, fibroblasts and endothelial cells. GM-CSF stimulates stem cells of granulocyte or macrophage to induce the differentiation thereof and proliferation of granulocyte or macrophage colonies. M-CSF and G-CSF primarily induce the formation of the colonies of macrophage and granulocyte, respectively. In vivo, G-CSF induces the differentiation of bone marrow leucocytes and enhances the function of mature granulocyte and, accordingly, it's clinical importance in treating leukemia has been well established.
Human G-CSF (hG-CSF) is a protein consisting of 174 or 177 amino acids, the 174 amino-acid variety having higher neutrophil-enhancing activity (Morishita, K. et al., J. Biol. Chem., 262, 15208-15213 (1987)). The amino acid sequence of hG-CSF consisting of 174 amino acids is shown in FIG. 1 and there have been many studies for the mass production of hG-CSF by manipulating a gene encoding said hG-CSF.
For instance, Chugai Pharmaceuticals Co., Ltd. (Japan) has disclosed the amino acid sequence of hG-CSF and a gene encoding same (Korean Patent Publication Nos. 91-5624 and 92-2312), and reported a method for preparing proteins having hG-CSF activity by a gene recombination process (Korean Patent Nos. 47178, 53723 and 57582). In this preparation method, glycosylated hG-CSF is produced in a mammalian cell by employing a genomic DNA or cDNA comprising a polynucleotide encoding hG-CSF. The glycosylated hG-CSF has an O-glycosidic sugar chain, but, it is known that said sugar chain is not necessary for the activity of hG-CSF (Lawrence, M. et al., Science, 232, 61 (1986)). Further, it is also well-known that the production of glycosylated hG-CSF employing mammalian cells requires expensive materials and facilities, and therefore, such a process is not economically feasible.
Meanwhile, there have been attempts to produce non-glycosylated hG-CSF by employing a microorganism, e.g., E. coli. In these studies, hG-CSFs having 175 or 178 amino acids having a methionine residue attached at the N-terminus thereof are obtained due to the ATG initiation codon employed in the microorganism. The additional methionine residue, however, causes undesirable immune responses in human body when the recombinant hG-CSF is administered thereto (European Patent Publication No. 256,843). Further, most of the methionine-containing hG-CSFs produced in E. coli are deposited in the cells as insoluble inclusion bodies, and they must be converted to an active form through a refolding process, at a significant loss of yield. In this regard, four of the five Cys residues present in wild-type hG-CSF participate in forming disulfide bonds, while the remaining one contributes to the aggregation of the hG-CSF product during the refolding process to lower the yield.
Recently, in order to solve the problems associated with the production of a foreign protein within a microbial cell, various efforts have been made to develop a method based on efficient secretion of a target protein across the microbial cell membrane into the extra-cellular domain.
For instance, in a method employing a signal peptide, a desired protein is expressed in the form of a fusion protein wherein a signal peptide is added to the N-terminus of the protein. When the fusion protein passes through the cell membrane, the signal peptide is removed by an enzyme and the desired protein is secreted in a mature form. The secretory production method is advantageous in that the produced amino acid sequence is usually identical to the wild-type. However, the yield of a secretory production method is often quite low due to unsatisfactory efficiencies in both the membrane transport and the subsequent purification process. This is in line with the well-known fact that the yield of a mammalian protein produced in a secretory mode in prokaryotes is very low: Hitherto, no microbial method has been reported for the efficient expression and secretion of soluble hG-CSF having no added methionine residue at its N-terminus.
The present inventors have previously reported the use of a new secretory signal peptide prepared by modifying the signal peptide of E. coli thermoresistant enterotoxin II (Korean Patent Laid-open publication No. 2000-19788) in the production of hG-CSF. Specifically, an expression vector comprising a hG-CSF gene attached to the 3′-end of the modified signal peptide of E. coli thermoresistant enterotoxin II was prepared, and biologically active, mature hG-CSF was expressed by employing E. coli transformed with the expression vector. However, most of the expressed hG-CSF accumulated in the cytoplasm rather than in the periplasm.
The present inventors have endeavored further to develop an efficient secretory method for the production of hG-CSF in a microorganism and have found that a modified hG-CSF, which is prepared by replacing at least one amino acid residue, especially, the 17th cysteine residue, of wild-type hG-CSF with other amino acid, retains the biological activity of the wild-type, and that the modified hG-CSF having no methionine residue at the N-terminus thereof can be efficiently expressed and secreted by a microorganism when an appropriate secretory signal peptide is employed.